Cell membranes consist of a double layer of lipid molecules in which various proteins are embedded. Because of its hydrophobic interior, the lipid bilayer of cell membranes serves as a barrier to the passage of most polar molecules and therefore is crucial to cell viability. To facilitate the transport of small water-soluble molecules into or out of cells or intracellular compartments, such membranes possess carrier and channel proteins. Ion channels are essential for many cellular functions, including the electrical excitability of muscle cells and electrical signaling in the nervous system (reviewed by Alberts et al., 1994). They are present not only in all animal and plant cells, as well as microorganisms, but also have been identified in viruses (Ewart et al., 1996; Piller et al., 1996; Pinto et al., 1992; Schubert et al., 1996; Sugrue et al., 1990; Sunstrom et al., 1996), where they are thought to play an important role in the viral life cycle.
The influenza A virus is an enveloped negative-strand virus with eight RNA segments encapsidated with nucleoprotein (NP) (reviewed by Lamb and Krug, 1996). Spanning the viral membrane are three proteins: hemagglutinin (HA), neuraminidase (NA), and M2. The extracellular domains (ectodomains) of HA and NA are quite variable, while the ectodomain domain of M2 is essentially invariant among influenza A viruses. The life cycle of viruses generally involves attachment to cell surface receptors, entry into the cell and uncoating of the viral nucleic acid, followed by replication of the viral genes inside the cell. After the synthesis of new copies of viral proteins and genes, these components assemble into progeny virus particles, which then exit the cell (reviewed by Roizman and Palese, 1996). Different viral proteins play a role in each of these steps. In influenza A viruses, the M2 protein which possesses ion channel activity (Pinto et al., 1992), is thought to function at an early state in the viral life cycle between host cell penetration and uncoating of viral RNA (Martin and Helenius, 1991; reviewed by Helenius, 1992; Sugrue et al., 1990). Once virions have undergone endocytosis, the virion-associated M2 ion channel, a homotetrameric helix bundle, is believed to permit protons to flow from the endoscopes into the virion interior to disrupt acid-labile M1protein-ribonucleoprotein complex (RNP) interactions, thereby promoting RNP release into the cytoplasm (reviewed by Helenius, 1992). In addition, among some influenza strains whose HAs are cleaved intracellularly (e.g., A/fowl plagues/Rostock/34), the M2 ion channel is thought to raise the pH of the trans-Golgi network, preventing conformational changes in the HA due to conditions of low pH in this compartment (Hay et al., 1985; Ohuchi et al., 1994; Takeuchi and Lamb, 1994).
Evidence that the M2 protein has ion channel activity was obtained by expressing the protein in oocytes of Xenopus laevis and measuring membrane currents (Pinto et al., 1992; Wang et al., 1993; Holsinger et al., 1994). Specific changes in the M2 protein transmembrane (TM) domain altered the kinetics and ion selectivity of the channel, providing strong evidence that the M2 TM domain constitutes the pore of the ion channel (Holsinger et al., 1994). In fact, the M2 TM domain itself can function as an ion channel (Duff and Ashley, 1992). M2 protein ion channel activity is thought to be essential in the life cycle of influenza viruses, because amantadine hydrochloride, which blocks M2 ion channel activity (Hay et al., 1993), inhibits viral replication (Kato and Eggers, 1969; Skehel et al., 1978).
The genome of influenza B virus, a member of the family Orthomyxoviridae, consists of eight negative-strand RNA segments, which encode 11 proteins. Of these, nine are also found in influenza A virus: three RNA-dependent RNA polymerase subunits (PB1, PB2, and PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (M1), and two nonstructural proteins (NS1 and NS2). Two proteins, NB and BM2 are unique to influenza B virus. NB is encoded by RNA segment 6, which also encodes NA, while BM2 is encoded by segment 7.
The NB protein of influenza B virus is a type III integral membrane protein, expressed abundantly on the surface of virus-infected cells (Betakova et al., 1996; Shaw et al., 1983; Shaw et al., 1984), and is incorporated into virions (Betakova et al., 1996; Brassard et al., 1996). This small protein (100 amino acids) possesses an 18-residue N-terminal ectodomain, a 22-residue transmembrane domain, and a 60-residue cytoplasmic tail (Betakova et al., 1996; Williams et al., 1986). From previous studies measuring membrane currents, and by analogy with the M2 protein of influenza A virus (Fisher et al., 2000; Fisher et al., 2001; Sunstrom et al., 1996), NB was thought to function as an ion channel protein. However, the electrophysiological measurements of NB protein based on the lipid bilayer system are difficult to interrupt. That is, proteins and peptides containing hydrophobic domains, which are believed to lack ion channel activity in cells, can yield channel recordings in lipid bilayers (Lear et al., 1988; Tosteson et al., 1988; Tosteson et al., 1989). Moreover, in the studies of Fischer et al. (2001), and Sunstrom et al. (1996), amantadine was used to demonstrate the loss of channel activity by NB protein, despite the inability of this drug to inhibit influenza B virus replication. Thus, the available evidence challenges the notion that the NB protein has ion channel activity.
Immunity to viral infections depends on the development of an immune response to antigens present on the surface of infected cell or on the virions. If the surface viral antigens are known, successful vaccines can be produced. Although there may be several antigens present on the surface, only some of them produce neutralizing immunity. One method to produce a vaccine is to “attenuate” the virus. This is usually done by passing infectious virus into a foreign host and identifying strains that are super virulent. Normally, these super virulent strains in the foreign host are less virulent in the original host cell, and so are good vaccine candidates as they produce a good immune response in the form of humoral IgG and local IgA.
Generally, influenza vaccines have been prepared from live, attenuated virus or killed virus which can grow to high titers. Live virus vaccines activate all phases of the immune system and stimulate an immune response to each of the protective antigens, which obviates difficulties in the selective destruction of protective antigens that may occur during preparation of inactivated vaccines. In addition, the immunity produced by live virus vaccines is generally more durable, more effective, and more cross-reactive than that induced by inactivated vaccines. Further, live virus vaccines are less costly to produce than inactivated virus vaccines. However, the mutations in attenuated virus are often ill-defined and those mutations appear to be in the viral antigen genes.
Thus, what is needed is a method to prepare recombinant attenuated influenza virus for vaccines e.g., attenuated viruses having defined mutation(s).